A key rationale of this project is the growing need for an improved low-cost, high sensitivity multigene mutation profiling method for tumors. An important example is the recent recommendation of the National Comprehensive Cancer Network (NCCN) to screen all patients diagnosed with metastatic colorectal cancer for KRAS mutations in the primary tumor or a site of metastasis. Mutations in KRAS and other genes downstream of EGFR have been found to be highly predictive of resistance to anti-EGFR therapies. A second example is the case of cancer recurrence, where mutations in several genes have been found to be highly correlated with metastatic relapse, although further research is still needed before the efficacy of particular multigene mutation panels are established. In general, a lower-cost, higher sensitivity method than conventional DNA sequencing capable of scanning for mutation in multiple genes from tumors including from FFPE blocks is needed to improve current CRC tumor screening and accelerate the development of new screening assays. During Phase I, a new method based on mass spectrometry of in vitro expressed proteins (MASSIVE-PRO) was developed for such a purpose. Initial studies demonstrated that as low as 1- 2% mutant population could be detected in three test genes: APC, KRAS and P53. Since MASSIVE-PRO is a scanning technique, only the WT sequence of a gene needs to be known in order to discover a new mutation which is added to the gene signature. During Phase II, research will focus on the improvement of MASSIVE-PRO and its application to molecular profiling of CRC tissue with particular focus on developing an assay to more accurately predict mCRC patient's resistance to anti-EGFR drugs. Advanced techniques for cell-free protein expression and mass spectrometry will be introduced to significantly improve the ability to detect and characterize mutations including the use of reconstituted extracts, primer design, MS-MS sequencing, isotope depletion, multiplex processing and detection and automation. We will obtain and analyze 200 tumor samples stored as FFPE blocks from CRC tissue archives along with correlated patient history in collaboration with Dr. Paul Schroy, Director of Clinical Research for the Section of Gastroenterology at the Boston Medical Center and Dr. Brent Zanke, Vice-President of the Ontario Institute for Cancer Research Tumor Bank. Choice of target genes and regions scanned is based on recent studies showing that mutations in KRAS, BRAF, PTEN and PIK3 result in resistance to anti-EGFR drug therapy. For the purpose of ultimate clinical implementation, regulatory (FDA) approval, and marketing of a molecular profiling assay based on MASSIVE-PRO AmberGen will work closely with both Genzyme Corporation and Quest Diagnostics, Inc., two of the largest diagnostic companies in the U.S. All results will be statistically analyzed in collaboration with Prof. Josie Dupuis, Associate Professor of Biostatistics at Boston University School of Public Health. PUBLIC HEALTH RELEVANCE: There exists an increasing need to develop a low-cost, high sensitivity method for scanning for mutations in the DNA from tumors. In the case of colorectal cancer, one example is the need to identify those patients who are resistant to anti-EGFR drugs. Current methods for mutation detection, such as conventional DNA sequencing are not sufficiently sensitive to be used routinely in clinical practice. This project aims to develop a novel low-cost, high-throughput method for profiling CRC tumors based on cell-free protein translation and mass spectrometry, which if successful will reduce the high mortality rate of the over 150,000 U.S. citizens diagnosed each year with CRC.